This section provides background information related to the present disclosure which is not necessarily prior art.
Traditional plant breeding strategies to develop new lines of plants that exhibit particular traits are time consuming and sometimes unpredictable. More recently, the development of methods for plant genetic transformation and the growing identification and availability of useful genes and their products has opened the door to rapid development of plants expressing desired traits. However, there remains a need for improved methods. Existing strategies, such as Agrobacterum—mediated transformation and particle bombardment depend heavily on the tissue and genotype. Cell penetrating peptides (CPPs) are a novel and fast growing class of short peptides that are known to play an important role in translocation of a wide range of cargo complexes including proteins and DNA across the bio-membranes in mammalian and human cell lines (Schwartz and Zhang, 2000; Langel, 2002; Vives, 2002,).
The HIV-1 TAT protein transduction domain (PTD) is one of the most well studied translocating peptides. Recent reports have shown the potential of TAT-PTD and its oligomers for plasmid delivery by forming a complex with the negatively charged DNA in mammalian cells (Ignatovich et al, 2003; Rudolph e al, 2003; Siprashvili et al, 2003; Hellgren et al, 2004). Other peptides that have been shown to have translocating properties include pVEC, transportan, penetratin, pep-1 peptides and fragments thereof.
Some of the prior art relating to CPP mediated translocation is discussed below. United States Patent Application 20040121325 describes a method of producing recombinant plants or plant cells which express a sequence coding for a protein having xylosyltransferase activity or being complementary thereto.
PCT Application WO2005117992 discloses a composition for controlled delivery of a compound into a target cell. The composition comprises a cell-penetrating peptide, a cell penetrating peptide inhibitor, a compound, and a cleavage site where the peptide inhibitor inhibits translocation activity of the cell penetrating peptide. Cleavage at the cleavage site by a cleaving agent disinhibits the cell penetrating peptide and the disinhibited cell penetrating peptide is capable of translocating a compound into a target cell. This application does not, however, disclose transformation of plant cells.
United States Patent Application No. 2005/0260756 discloses a membrane permeable complex for facilitating delivery of a double-stranded RNA molecule into a cell. The complex comprises a double-stranded RNA molecule and a cell-penetrating peptide with a covalent bond linking the double-stranded RNA to the cell penetrating peptide. The disclosure is limited to the transformation of neuronal cells.
Unnamalai et al. (FEBS Letters 566 (2004) 307) disclose the use of a cationic oligopeptide polyarginine for delivery of dsRNA for post-transcriptional gene silencing.
While CPPs have been shown to facilitate cargo delivery in mammalian cells, the use of CPP in plant cells for transfection studies has been limited by a number of factors. A major obstacle to adapting this technology to plants is that, unlike animal cells, plant cells present a dual barrier system (cell wall and plasma membrane) for the internalization of CPPs and their cargos. Therefore, CPPs must overcome these two barriers for efficient translocation.
With the ever-growing information from the plant genome-sequencing projects there is an urgent need for the development of a fast, universal (tissue/genotype independent) method in plants for functional genomic studies of a wide array of genes and for the development of transgenic plants expressing desired traits.